In the healthy human body the rate of red blood cell production is controlled by the hypoxia-regulated expression of erythropoietin (EPO). Patients suffering from various chronic diseases often develop anemia that can be alleviated by EPO replacement therapy. Currently, exogenous EPO is provided as a frequently administered recombinant protein. An attractive alternative would be to deliver the EPO gene into ectopic sites of the body to provide an internal hormone source for the long-term management of a chronic disease. For the transition of an EPO-based gene therapy to the clinic, tools to regulate expression levels would be required to maintain red blood cell counts in the normal range. Most currently available drug-dependent gene regulatory systems rely on protein factors that are foreign to the human body. To avoid the risk of immune rejection triggered by such foreign proteins, we plan to create a protein-free system for the drug-dependent regulation of expression from exogenous EPO genes delivered into the patient's limb muscles. The system will be based on inserting a drug-sensing riboswitch into the EPO mRNA that can turn expression on when the patient is taking the drug-ligand. The major goals of this Phase I project are: (1) to use a randomized pool of ssRNAs to select for aptamers that undergo ligand-dependent conformational changes, and (2) to insert them into EPO expression cassettes in order to assess their ability to regulate EPO expression in vitro, in transiently transfected cultured cells. Our Phase I efforts should provide proof of concept that the selected riboswitch can serve as a functional, drug-dependent regulator of expression levels in the context of the full-length EPO mRNA, in live cells. Phase II efforts will test the most promising regulated EPO expression construct(s) in vivo, initially in mice, followed by studies in non-human primates. The overall goal is the stable maintenance of a desired level of erythropoiesis by administering or withdrawing the inducer drug, as indicated by changes in the patient's hematological parameters. This gene therapy would provide a safe, long-term treatment option for patients with chronic anemia. The drug-sensing riboswitch can also be incorporated into expression cassettes encoding other therapeutic proteins, such as insulin and other hormones, growth factors or therapeutic antibodies. PUBLIC HEALTH RELEVANCE: Recombinant human EPO protein currently represents Medicare's highest drug expense. Gene therapy is a promising, more economical alternative for the long-term management of chronic anemia. However, since not only the lack, but also the overexpression of EPO can cause serious health problems, the cornerstone of developing safe gene-based therapy is finding a reliable tool for regulating expression. Our proposed non- immunogenic regulatory system could control EPO expression by taking a drug that is already FDA-approved for long-term use in human patients. [unreadable] [unreadable] [unreadable]